Identification of N-Acetyltransferase 2 Genotypes by Continuous Monitoring of Fluorogenic Hybridization Probes

University Hospital RWTH Aachen, Aachen, North Rhine-Westphalia, Germany
Analytical Biochemistry (Impact Factor: 2.22). 12/1999; 275(1):93-7. DOI: 10.1006/abio.1999.4288
Source: PubMed


Three polymorphic sites in the N-acetyltransferase 2 (NAT2) gene were detected using rapid cycle DNA amplification with allele-specific fluorescent probes and melting curve analysis. Two fluorogenic adjacent hybridization probes were designed to NAT2*5A (C(481)T), NAT2*6A (G(590)A), and NAT2*7A (G(857)A). During amplification, probe hybridization is observed as fluorescence resonance energy transfer. The fluorescence increases every cycle as the product accumulates during amplification. A single base mismatch resulted in a melting temperature shift (T(m)) of 5 to 6 degrees C, allowing for the easy distinction of a wild-type allele from the mutant allele. The protocol is rapid, requiring 40 min for the completion of 45 cycles including the melting curves. It is also a simple and flexible method, since DNA templates prepared from different sources, including DNA from serum and paraffin-embedded tissue sections, could be used without adverse effects. Fluorescence genotyping of all three polymorphisms in a total of 155 DNA samples correlated perfectly with our previously validated genotyping by restriction enzyme digestion (PCR-RFLP). This new facile approach allows for the easy detection of NAT2 polymorphisms in hundreds of samples in only a day.

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    • "These methods can be convenient, but require an additional step for end point detection. Analytical methods that reduce sample processing to a single-step procedure were also developed, including nick-translation PCR (17), hybridization with FRET probes (18), melting curves analysis (19–22), fluorogenic allele-specific PCR (23), universal FRET reagents in a single-step Invader assay (24), universal TaqMan probes (25) and others. However, several of these methods cannot deal with detection of a low concentration of mutant target in the presence of a large excess of wild-type DNA that is often found in clinical specimens. "
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    ABSTRACT: Real-time signal generation methods for detection and characterization of low-abundance mutations in genomic DNA are powerful tools for cancer diagnosis and prognosis. Mutations in codon 12 of the oncogene KRAS, for example, are frequently found in several types of human cancers. We have developed a novel real-time PCR technology, FLAG (FLuorescent Amplicon Generation) and adapted it for simultaneously (i) amplifying mutated codon 12 KRAS sequences, (ii) monitoring in real-time the amplification and (iii) genotyping the exact nucleotide alteration. FLAG utilizes the exceptionally thermostable endonuclease PspGI for real-time signal generation by cleavage of quenched fluorophores from the 5′-end of the PCR products and, concurrently, for selecting KRAS mutations over wild type. By including peptide-nucleic-acid probes in the reaction, simultaneous genotyping is achieved that circumvents the requirement for sequencing. FLAG enables high-throughput, closed-tube KRAS mutation detection down to ∼0.1% mutant-to-wild type. The assay was validated on model systems and compared with allele-specific PCR sequencing for screening 27 cancer specimens. Diverse applications of FLAG for real-time PCR or genotyping applications in cancer, virology or infectious diseases are envisioned.
    Full-text · Article · Feb 2007 · Nucleic Acids Research
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    • "Promoter fragments containing more than two SNPs were genotyped by direct sequencing. All other SNPs were genotyped with the LightCycler™ assay (Roche, Mannheim, Germany) based on hybridization of probes labeled by two different dyes allowing Fluorescence Resonance Energy Transfer (FRET) [33]. A genotyping quality control was performed by introducing duplicates in the PCR plates and by genotyping all individuals twice. "
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    Full-text · Article · Apr 2005 · BMC Genetics
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    • "It should be noted that the study presented here is preliminary and its statistical power is limited due to the relatively small number of samples analysed, particularly the study of NAT2 genotype and risk of AD. Therefore much larger case-control investigations, using more highly automated detection methods such as the LightCycler real-time PCR methods of Blömeke et al. [38] and Wikman et al. [39] will help to validate the results shown here. In order to detect a 1.5 fold increased risk of AD associated with the NAT1*10 allele, approximately 650 cases and 650 controls would need to be studied to give 80% power of achieving significance at the 5% level. "
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